Production of poly-?-hydroxybutyrate by Alcaligenes eutrophus transformants harbouring cloned phbCAB genes

Park, Jin Seo; Park, Hae Chul; Huh, Tae Lin; Lee, Yong Hyun

doi:10.1007/bf00130360

Cited by 45 publications

(14 citation statements)

References 9 publications

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“…Using transformed R. eutropha strains where either the PHB synthase or the PhbA ;-ketothiolase and acetoacetylCoA reductase had been overexpressed, these investigators found that enhanced levels of PHB synthase signifi cantly increased the molar fractions of 3HV and 4HB under conditions where P(3HB-3HV) and P(3HB-4HB) were produced. Park et al (1995cPark et al ( , 1997 reported that PHB synthase was the main controlling enzyme for PHB production in transformed R. eutropha strains. This differs somewhat with our predictions for plant production under dark conditions, but from the flux control coefficients calculated under light conditions, it is quite evident that about 400 of the control lies with PHB synthase.…”

Section: Resultsmentioning

confidence: 98%

Metabolic Modeling as a Tool for Evaluating Polyhydroxyalkanoate Copolymer Production in Plants

Daae

Dunnill

Mitsky

et al. 1999

Metabolic Engineering

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Section: Resultsmentioning

confidence: 98%

Metabolic Modeling as a Tool for Evaluating Polyhydroxyalkanoate Copolymer Production in Plants

Daae

Dunnill

Mitsky

et al. 1999

Metabolic Engineering

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“…The small arrowheads denote an internal standard, the protein tropomyosin (MW, 33,000; pI 5.2). (25), so cobalt was added to the glucose feed. During the initial induction phase, the phosphate concentration rapidly decreased in the reactor (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…E. coli strains were grown in Luria-Bertani medium. R. eutropha strains were cultivated in one of the following media depending on the application: NR medium (36), PCT medium (Table 2), and Lee medium (25). NR is a complex medium, while both PCT and Lee media are minimal media.…”

Section: Methodsmentioning

confidence: 99%

A Novel High-Cell-Density Protein Expression System Based on Ralstonia eutropha

Srinivasan

Barnard

Gerngross

2002

Appl Environ Microbiol

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We describe the development of a novel protein expression system based on the industrial fermentation organism Ralstonia eutropha (formerly known as Alcaligenes eutrophus) NCIMB 40124. This new system overcomes some of the shortcomings of traditional Escherichia coli-based protein expression systems, particularly the propensity of such systems to form inclusion bodies during high-level expression. Using a proteomics approach, we identified promoters that can be induced by simple process parameters or medium compositions in high-density cell culture or shake flasks, respectively. By combining newly developed molecular biological tools with a high-cell-density fermentation process, we were able to produce high levels (>1 g/liter) of soluble, active organophosphohydrolase, a model enzyme prone to inclusion body formation in E. coli.Advances in protein engineering, the completion of numerous bacterial and fungal genome sequencing projects, and the isolation of new genes from extremophiles have led to an increased number of useful proteins. However, to enable recombinant proteins to play a role in applications where larger quantities are required, such as tissue engineering or catalytic materials, production technologies that are more efficient and robust are desirable. Improving protein production is the primary goal of recombinant microbial process development and is a focus of our laboratory. Overall protein productivity can be improved by increasing the product of two variables: (i) the amount of recombinant protein per cell (specific productivity) and (ii) the amount of cell mass per unit of volume and time (cell productivity). In order to improve volumetric productivity in a cost-effective manner, recombinant proteins are often produced in high-cell-density fermentations. High-cell-density fermentations offer many advantages over traditional fermentations in that final product concentrations are higher and downtime and water usage are reduced, yet overall productivity is improved, resulting in lower setup and operating costs (3, 6).In the absence of specific folding or posttranslational modification requirements, Escherichia coli is usually the expression host of choice. E. coli-based fermentation systems produce good yields at laboratory scale; however, scale up to industrial scale, where oxygen enrichment, dialysis, and sophisticated feeding algorithms become impractical and cost prohibitive, has been challenging (22). Unlike laboratory-scale fermentors, where the above-mentioned techniques are feasible, largescale industrial fermentors are limited by mixing constraints and their ability to transfer oxygen and heat. It is well documented that oxygen-limiting conditions in E. coli result in the production of reduced carbon metabolites such as acetate, lactate, and formate (1, 20), which accumulate in high-celldensity cultures and ultimately inhibit further microbial growth. Titers of recombinant proteins in E. coli are limited by several factors including the final cell density (22), the tendency for inclusion...

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“…Moreover, the key enzymes of PHA biosynthesis (PHA synthases) are well known for their low substrate specificity, which is responsible for the large number of different PHA constituents found in microbial PHAs (59). As in the case of PHB synthases, different PHA synthases were cloned and expressed in various prokaryotic organisms (46,47,55,58,67); eukaryotic organisms, such as Saccharomyces cerevisiae (40); insect cells (73); and plants (21,48,53). Recently, the range of polymers synthesized by PHA synthases was extended to polythioesters consisting of 3-mercaptoalkanoates by employing PHA-accumulating bacteria, such as Cupriavidus necator (formerly Ralstonia eutropha), which typically accumulate PHB and other short-chain-length PHA, or engineered Escherichia coli strains and various organic thiochemicals as precursor substrates (42,43).…”

mentioning

confidence: 99%

Large-Scale Production of Poly(3-Hydroxyoctanoic Acid) by Pseudomonas putida GPo1 and a Simplified Downstream Process

Elbahloul

Steinbüchel

2009

Appl Environ Microbiol

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The suitability of Pseudomonas putida GPo1 for large-scale cultivation and production of poly(3-hydroxyoctanoate) (PHO) was investigated in this study. Three fed-batch cultivations of P. putida GPo1 at the 350-or 400-liter scale in a bioreactor with a capacity of 650 liters were done in mineral salts medium containing initially 20 mM sodium octanoate as the carbon source. The feeding solution included ammonium octanoate, which was fed at a relatively low concentration to promote PHO accumulation under nitrogen-limited conditions. During cultivation, the pH was regulated by addition of NaOH, NH 4 OH, or octanoic acid, which was used as an additional carbon source. Partial O 2 pressure (pO 2 ) was adjusted to 20 to 40% by controlling the airflow and stirrer speed. Under the optimized conditions, P. putida GPo1 was able to grow to cell densities as high as 18, 37, and 53 g cells (dry mass) (CDM) per liter containing 49, 55, and 60% (wt/wt) of PHO, respectively. The resulting 40 kg CDM from these three cultivations was used directly for extraction of PHO. Three different methods of extraction of PHO were applied. From these, only acetone extraction showed better performance and resulted in 94% recovery of the PHO contents of cells. A novel mixture of precipitation solvents composed of 70% (vol/vol) methanol and 70% (vol/vol) ethanol was identified in this study. The ratio of PHO concentrate to the mixture was 0.2:1 (vol/vol) and allowed complete precipitation of PHO as white flakes. However, at a ratio of 1:1 (vol/vol) of the solvent mixture to PHO concentrate, a highly purified PHO was obtained. Precipitation yielded a dough-like polymeric material which was cast into thin layers and then shredded into small strips to allow evaporation of the remaining solvents. Gas chromatographic analysis revealed a purity of about 99% ؎ 0.2% (wt/wt) of the polymer, which consisted mainly of 3-hydroxyoctanoic acid (96 mol%).

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Production of poly-?-hydroxybutyrate by Alcaligenes eutrophus transformants harbouring cloned phbCAB genes

Cited by 45 publications

References 9 publications

Metabolic Modeling as a Tool for Evaluating Polyhydroxyalkanoate Copolymer Production in Plants

Metabolic Modeling as a Tool for Evaluating Polyhydroxyalkanoate Copolymer Production in Plants

A Novel High-Cell-Density Protein Expression System Based on Ralstonia eutropha

Large-Scale Production of Poly(3-Hydroxyoctanoic Acid) by Pseudomonas putida GPo1 and a Simplified Downstream Process

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